The long-term goal of the proposed research is to develop a simple quantitative point-of-care (POC) method for determination of percentage of circulating CD4+ T-lymphocytes and CD4/CD8 ratio in blood of pediatric patients. CD4 cell counts are needed to stage and monitor HIV-infected patients. In adults, the absolute number of CD4+ T-cells per microlitre of blood has critical prognostic and therapeutic implications and is used for both HIV staging and treatment decisions. In pediatric patients, the high variability of absolute CD4+ T-cell numbers occurs within first 5 or 6 years of age. In addition, concurrent illnesses may affect CD4 counts. Therefore, in pediatric patients aged less than 5 or 6 years, it is strongly recommended that percentage of CD4+ T-cell measurement within the lymphocyte populations (%CD4/ly) is determined since this parameter (%CD4/ly) is far less variable than the absolute CD4+ T-cell counts. The gold standard technique currently used for quantitation of CD4 cells and determination of %CD4/ly is flow cytometry. However, flow cytometers, including smaller single-purpose CD4 cell counting devices, are expensive and are generally inaccessible in resource-limited countries. Because flow cytometry-based CD4 cell counting is beyond the reach of so many HIV-infected people, the World Health Organization (WHO) has urged the development of simple point-of-care CD4 cell count and %CD4/ly assays designed for resource- limited settings. Recently, a number of manual assays have emerged to meet specific demands for rapid testing of CD4 cell count in blood. However, significant limitations of these assays, such as multi-step operating procedures, use of relatively expensive equipment and slow sample turnover hinder their widespread use. Moreover, to our knowledge currently there are no manual methods on the market that provide %CD4/ly measurements. It is in this context that we propose to develop a quantitative, fast and inexpensive assay for determination of percentage of CD4+ T-cells in less than 5 years old children. The proposed method, flow-through cell counting assay (FTCA) is based on the use of the special membrane and a proprietary flow-through cassette to measure cell count status. The test can potentially be completed in less than 15 minutes and can be performed by laboratory technicians with low levels of training. The specific aims of Phase I are: 1) Development of an a-prototype of the disposable FTCA cartridge for simultaneous detection of CD4+, CD8+ T-cells and total lymphocytes; 2) Examine and select the most suitable antibodies and filters for determination of CD4, CD8 counts and total lymphocytes by FTCA. Explore all variables affecting the performance of the FTCA and optimize the conditions of the assay; 3) Compare the analytical performance of %CD4/ly and CD4/CD8 determination by the FTCA and conventional flow cytometry assay using a limited number of well-characterized clinical samples. Advantages of the proposed technology such as the speed, convenience, and low cost of the analysis substantiates our belief that the FTCA will be widely used in clinical practice.